Comparative genomic hybridisation (CGH) is a molecular-cytogenetic method for the analysis of copy number changes (gains/losses) in a subject's DNA. The technique relies on the comparison of two labelled samples by allowing them to hybridise and subsequently looking for regions of differential hybridisation. It has been used particularly in cytogenetic analysis, where it allows the comparison of a genome isolated from a clinical (test) sample (e.g. derived from a cancer patient) with a control (reference) sample in a single hybridisation. It was originally disclosed in reference 1.
Whereas early CGH methods relied on hybridisation to a reference chromosome sample, array-based CGH methods have since been developed [2-4]. In these methods, the reference chromosome is replaced by an array of immobilised nucleic acid probes, with the individual immobilised sequences having known chromosomal locations and covering the genome to a desired degree. By choosing appropriate probes, this method gives the potential to cover any genomic region of interest, and to any desired resolution. Commercial array CGH kits are now available, including Spectral Chip from Perkin Elmer, CytoChip from BlueGnome, and CGH products from Nimblegen.
These two distinct methods are referred to as ‘chromosomal CGH’ and ‘array CGH’.
Copy number variation in humans can result in certain disease types and, although CGH is a powerful tool for analysing copy number changes in a given subject's DNA, it is only able to provide information about unbalanced chromosomal changes. Structural chromosome aberrations such as balanced reciprocal translocations or inversions can not be detected, as they do not change the copy number, nor is it able to detect copy number neutral loss of heterozygosity (LOH) due to uniparental disomy (UPD).
UPD occurs when two copies of a chromosome, or part of a chromosome, are inherited from one parent and no copies from the other parent [5]. When the (two) homologous chromosomes are inherited from one parent, this is called a heterodisomic UPD. Heterodisomy indicates a meiosis I error. When the two (identical) replica copies of a single homolog of a chromosome are inherited, this is called an isodisomic UPD. Isodisomy indicates either a meiosis II error or postzygotic duplication.
Most occurrences of UPD result in no phenotypical anomalies. However, isodisomy can lead to the manifestation of rare recessive disorders, for example Silver-Russell or Prader Willi syndromes.
Determining if UPD has occurred can use single-nucleotide polymorphisms (SNPs) as markers to track the chromosome and determine if LOH has occurred. A SNP is a DNA sequence variation occurring when a single nucleotide—A, T, C, or G—in the genome differs between members of a population. For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide at a given position. In this case there are two alleles: C and T. Almost all common SNPs have only two variants. Within a population, SNPs can be assigned an allele frequency which indicates the percentage of the population which possess a particular nucleotide residue at the SNP position.
Standard arrays for array CGH are not capable of detecting SNPs. Array CGH typically uses longer oligos and therefore tolerates a higher stringency to achieve an optimal copy number variation result. SNP arrays tend to use shorter oligos under lower stringency and therefore the standard SNP arrays can, in theory, be used to obtain copy number variation (CNV) data, but because they are not typically capable of generating data of a comparable quality to array CGH they are not used for this purpose. Thus, for example, reference 6 used two different arrays for its combined array-CGH and SNP-LOH analysis.
Reference 7 discloses arrays which can be used for both array CGH and SNP analysis. SNPs are detected by allele-specific chain extension of hybridised probes.
It is an object of the invention to provide methods and apparatuses for simultaneous array CGH and SNP array analysis. In particular it is an object of the invention to provide improved ways of distinguishing if LOH at a locus is caused by deletion or isodisomy.